The generation of physical maps and overlapping clone sets for the human genome has progressed to the point where contigs of YAC and cosmid clones will be available for most genomic regions within the next few years. The genetic map has also improved so that it is now relatively straightforward to localize disease genes by family studies. These two genome mapping efforts converge-at the level of identifying genes on the physical 'nap. Gene identification can help set priorities for large-scale DNA sequencing, can provide starting points and guideposts to aid in sequencing strategies and allows direct comparison with the genomes of model organisms. For disease gene identification, the driving force of much human genetic research, isolation of positionally implicated candidates is essential to discovering the culprit defect. The saturation of a particular chromosomal region with gene sequences for either or both of the above purposes is not efficiently achieved using a global genome mapping strategy. Rather, it requires the intensive application of gene identification -techniques to the particular region of interest. Presently, there are a number of different strategies which have been proposed to accomplish this task, but these have not been systematically compared. One of the most intensively analyzed regions of the human genome is band 4p16.3, site of the recently cloned Huntington's disease gene. Detailed genetic and physical maps are available, YAC and cosmid contigs have been constructed, and complete DNA sequence of a 2Mb segment is currently being generated. We propose to use this region to perform a much-needed assessment of strategies for saturating a genomic region with gene sequences and for detailed comparison of the homologous region in the mouse. We will compare exon amplification, cDNA selection based on affinity labeling, and sequence-based exon prediction as means of isolating expressed sequences, while thereby saturating this segment of 4p16.3 with genes. The equivalent region of the mouse genome will also be cloned and the content, orientation and genomic coverage of genes will be compared with man. This study should provide valuable experience for applying saturation strategies to any specified genomic region for the isolation of disease genes, for facilitating more detailed mapping strategies, or for making structural associations within or between species.